PROJECT TITLE: Chemical Biology Studies of the Dynamics and Inhibition of Peptidoglycan Biosynthesis PROJECT SUMMARY This MIRA application represents the fusion of two complementary research programs that are, broadly described, directed at the urgent public health threat posed by antibiotic resistance. Infectious diseases are the leading cause of death world-wide; unfortunately, antibiotic use provides a strong selective pressure that results in the selection of strains that are resistant to the antibiotic shortly after its deployment as a therapeutic. While the selection of bacterial resistance to new antibiotics is inevitable, the development of new antibiotics and/or the identification of new antibacterial targets is essential to stay ahead in our arms race with bacteria. While antibacterial agents have been developed against multiple bacterial targets, the best target for antibacterial development has been, and continues to be, the bacterial cell wall. This MIRA application will capitalize on our discovery of fluorescent D-amino acids (FDAAs) that have provided unprecedented and heretofore unavailable tools for the visualization of bacterial cell wall peptidoglycan (PG) dynamics in real time and in live bacterial cells. Specifically, we propose additional studies to elucidate the details of bacterial cell division and cell separation in Bacillus subtilis, and we will develop ?turn-on? probes that will enable the study of PG synthesis and dynamics in real-time and in live bacterial cells. We will also continue our effort directed at the synthesis and mechanistic study of cyclic depsipeptide antibiotics that inhibit PG biosynthesis. Furthermore, we have recently uncovered data that suggest the cyclic depsipeptides may have a second mechanism of action; specifically, inhibition of lipid recycling, an essential activity in the PG biosynthesis pathway. The lipid recycling pathway remains to be clearly elucidated and, when coupled with dual-mode activity that may be inherent to these cyclic depsipeptides under study, very promising new avenues for the identification of new antibacterial targets and development of new antibacterial agents are likely to emerge from this research effort.